The process of sequencing has reached an industrial scale through the application of automation, particularly in the form of robots and handling of small volumes of liquid in multiplexed formats. The process typically involves fragmentation of a genome, insertion of fragments of interest into a cloning vector, isolation of individual clones, purification of the vector containing the inserted fragment and using that inserted fragment as a template in a sequencing reaction. The sequence data obtained is then aligned using software to obtain contiguous sequence from the numerous fragments. This process is described in more detail below.
Different cloning vectors can be used to clone fragments, depending on the size of the fragment. The purpose of cloning is to ensure replication of the insert to give large numbers of copies through a biological system (bacterium or virus). Large fragments are often cloned into BACs (Bacterial Artificial Chromosomes) or cosmids. Smaller fragments are commonly cloned either in bacterial plasmids such as pUC18 or in the phage M13.
A typical process for de novo sequencing of a genome using fragments cloned into a plasmid involves a number of possible steps performed sequentially. Examples of these steps are broadly described as follows:
1. Preparation of Bacterial Cultures
Fragments of the genome in question are created and inserted into plasmids (for example pUC18) that are maintained in a strain of the bacterium Escherichia coli. This process is termed transformation.
The transformed bacteria are spread out on an agar plate containing growth medium and an antibiotic to select for those bacteria that contain the plasmid (which bears a gene that confers antibiotic resistance to the host bacterium). The agar plate may also include an indicator that specifically shows the presence of bacteria containing plasmids that contain the insert—i.e. not just a clone containing an ‘empty’ or insert-free plasmid. The bacterial culture is diluted prior to being spread out to such an extent that individual bacterial cells, and hence their daughter colonies, are likely to be well separated from each other on the plate. This ensures that individual colonies are picked which in turn contain clones of only one sequence.
The plates are incubated overnight at 37° C. Individual bacterial cells give rise to colonies of cells that should not overlap on the plate.
Colonies are picked up either manually or by robot and may be used directly to prepare plasmids or, more commonly, to seed an over night liquid culture (typically 1-2 ml) to obtain larger amounts of bacteria and thus large numbers of copies of the insert.
2. Isolation of Plasmid Containing the Insert
The quality of the template nucleic acid is a key factor for success in a sequencing reaction. The template may be a plasmid or a polymerase chain reaction (PCR) product prepared from a plasmid. While there are reports of direct sequencing of bacterial extracts (see, for example, Frothingliam, R., R. L. Allen, et al. (1991). “Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.” Biotechniques 11(1):40-4.; Chen, Q., C. Neville, et al. (1996)), most major sequencing facilities take great care to isolate pure plasmid in order to ensure sequencing success.
Many methods for plasmid isolation/purification have been developed. One common method is to (i) lyse bacterial cells using NaOH, (ii) precipitate protein and chromosomal DNA, (iii) isolate the plasmid in solution on glass matrix (or other purification column which selectively retains the nucleic acid to be isolated by adsorption or absorption) in the presence of a chaotrope such as guanidinium isothiocyanate (see for example U.S. Pat. No. 5,234,809) or sodium iodide, (iv) washing with an ethanolic solution to remove salts and other residual contaminants, and finally (v) elute the plasmid from the matrix with a low ionic strength buffer or water. The process also includes exposure to RNase to degrade RNA.
This method is the basis for a number of commercially available kits such as GFX Micro Plasmid Prep Kit (Amersham Pharmacia Biotech). These kits typically include a series of solutions, Solutions I, II and III wherein Solution I comprises approximately 100 mM Tris-HCl, pH 7.5, 10 mM EDTA, 400 μg/mL RNase I, Solution II comprises approximately 100 mM NaOH, 1% w/v SDS and Solution III comprises a buffered solution containing acetate and a chaotrope.
Modifications to the surface of the glass in the glass matrix are described, for example, in U.S. Pat. No. 5,606,046. Alternative methods include isolation by reversible, non-specific binding to magnetic beads in the presence of PEG and salt described, for example, in Hawkins, T. L., T. O'Connor-Morin, et al. (1994). “DNA purification and isolation using a solid-phase.” Nucleic Acids Res 22(21): 4543-4, or by triplex-mediated affinity capture (U.S. Pat. No. 5,591,841). Suitable purification materials include gels, resins, membranes, glass or any other surface which selectively retains nucleic acids.
Plasmid quality can then be assessed using agarose gel electrophoresis and the quantity can be determined spectrophotometrically, both techniques being familiar to those skilled in the art. It is advantageous to obtain plasmid in water or dilute buffer that is compatible with the subsequent step (i.e. PCR or a direct sequencing reaction such as cycle sequencing).
If plasmid yield (or possible quality) is insufficient for direct sequencing then a specific region of the plasmid covering the insert and flanking sequence can be amplified by a conventional polymerase chain reaction (PCR) to give a sequencing template. This PCR product must be ‘cleaned-up’ before being used as template for sequencing. Clean up includes removal of unincorporated nucleotides and primers that would otherwise disturb the cycle sequencing reaction. One method involves exposure to exonuclease III and shrimp alkaline phosphatase, killing these enzymes by heat denaturation and using the reaction directly in cycle sequencing.
3. Cycle Sequencing
A cycle sequencing reaction involves mixing template nucleic acid with sequencing primers, a thermostable DNA polymerase enzyme and a mixture of the four deoxynucleotides (dATP, dCTP, dGTP and dTTP) including a small proportion of one base in a dideoxy (chain terminating) form, followed by cycles of heating and cooling (i.e. thermocycling). The reaction is run either in four different tubes—each containing having small amounts of each of the dideoxynucleotides, together with a fluorescently-labelled primer—or in one tube through the use of dideoxynucleotides with different fluorescent labels and unlabelled primer. The result is a fluorescently-labelled ladder of nucleic acid chains complementing the sequence of the template strand. Cycle sequencing reactions are commonly run in a scale of 10-20 μl in a microtitre plate (96-well or 384-well) in a thermocycler.
4. Clean Up
Where labelled nucleotide terminators are used, the reaction mixture should be ‘cleaned up’ afterwards in order to remove unincorporated fluorescent nucleotides. These would otherwise appear in the electrophoretic separation of the sequencing ladder and reduce the quality of the results.
In the case when capillary electrophoresis machines are used, it is also necessary to remove salts from the sequencing ladders in order to facilitate electrokinetic injection. This clean up is generally performed either by precipitation by addition of ethanol and salt, or by gel filtration. In the case of primer-labelled reactions, desalting is necessary only if capillary electrophoresis is to be used.
5. Analysis of Sequencing Reaction
The stopped sequencing reaction is then separated in a denaturing gel which may either be a slab-gel (as for example used in the ABI PRISM 377 or ALFexpress) or in capillary columns (as for example MegaBACE (Amersham Pharmacia Biotech) or PE ABI 3700 (PE Biosystems)) for subsequent analysis.
More recently, automation of these steps has been described with an emphasis on microfabrication i.e. performing these steps in as small volumes as possible.
The majority of automation efforts have aimed at the use of robots to carry out the steps normally done manually using pipettors and microtitre plates (96-well and 384-well). The individual steps are done in separate plates and liquid transfers between plates and formats are done by pipetting robots. More recently, attempts have been made to integrate various steps in one device, albeit comprised of a number of robots. One example is the Sequatron developed by Trevor Hawkins (Whitehead Institute). This consists of robots to purify M13 and carry out sequencing reactions in preparation for separation in ultra thin slab gels or capillaries. The technology is based on solid-phase isolation of DNA (see, for example, Hawkins, T. L., T. O'Connor-Morin, et al. (1994). “DNA purification and isolation using a solid-phase.” Nucleic Acids Res 22(21): 4543-4) and makes possible throughputs in excess of 25,000 samples per 24 hours. In addition, a group at Washington University has developed robots for picking M13 plaques and template preparation again based on large robots and multititre plates.
Methods for isolation of DNA in the presence of chaotropes on micromachined silicon structures have been published (Christel, L. A., K. Petersen, et al. (1999). “Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration.” J Biomech Eng 121(1):22-7). U.S. Pat. No. 5,882,496, describes the fabrication and use of porous silicon structures to increase surface area of heated reaction chambers, electrophoresis devices, and thermopneumatic sensor-actuators, chemical preconcentrates, and filtering or control flow devices. In particular, such high surface area or specific pore size porous silicon structures will be useful in significantly augmenting the adsorption, vaporization, desorption, condensation and flow of liquids and gasses in applications that use such processes on a miniature scale.
Methods for direct sequencing of plasmids from single bacterial colonies in fused-silica capillaries have been developed (Zhang, Y., H. Tan, et al. (1999). “Multiplexed automated DNA sequencing directly from single bacterial colonies.” Analytical Chemistry 71(22): 5018-25). Alternative methods involve separation in glass chips including detection methods based on laser-excited confocal microscopy (see, for example, Kheterpal, I. and R. A. Mathies (1999). “Capillary array electrophoresis DNA sequencing.” Analytical Chemistry 71(1): 31A-37A).
U.S. Pat. No. 5,610,074 describes a centrifugal rotor for the isolation, in a sequence of steps, of a substance from a mixture of substances dissolved, suspended or dispersed in a sample liquid. Multiple samples are processed simultaneously by means of a plurality of fractionation cells, each of which contains a series of interconnected, chambered and vented compartments in which individual steps of the fractionation and isolation procedure take place. In this centrifugal rotor, the specific compartment occupied by the sample liquid or one of its fractions at any stage of the process is governed by a combination both the speed and direction of rotation of the rotor and gravitational force. The interconnections, chambers and passages of each compartment are sized and angled to prevent predetermined amounts of sample and reagent liquids from overflowing the compartment. However, such a rotor is relatively bulky, requires relatively large volumes of solutions and is complicated to manufacture.
Micro-analysis systems that are based on microchannels formed in a rotatable, usually plastic, disc, are often called a “centrifugal rotor”, “lab on a chip” or “CD devices”. Such discs can be used to perform analysis and separation of small quantities of fluids. The principle of moving liquids through channels in a plastic disc for the purpose of carrying out enzymatic assays is described, for example, in Duffy, D. C., H. L. Gillis, et al. (1999). “Microfabricated centrifugal microfluidic systems characterization and multiple enzymatic assays.” Analytical Chemistry 71(20): 4669-4678. One type of suitable plastic disc is those referred to as compact discs or CDs.
When such discs are rotated a centripetal force is directed towards the centre of the disc. Where fluid is in the disc, this centripetal force can be the result of several forces including surface tension, tensile forces and capillary force. Movement of fluids towards the outer diameter of the disc is achieved by overcoming the centripetal force, usually by increasing the rotational speed of the disc.
In order to reduce costs it is desirable that the discs should be not restricted to use with just one type of reagent or fluid but should be able to work with a variety of fluids. Furthermore it is often desirable during the preparation of samples that the disc permits the user to dispense accurate volumes of any desired combination of fluids or samples without modifying the disc. Due to the small widths of the microchannels, any air bubbles present between two samples of fluids in the microchannels can act as separation barriers or can block the microchannel and thereby can prevent a fluid from entering a microchannel that it is supposed to enter. In order to overcome this problem U.S. Pat. No. 5,591,643 teaches the use of a centrifugal rotor which has microchannels that have cross sectional areas which are sufficiently large that unwanted air can be vented out of the microchannel at the same time as the fluid enters the microchannel.